The invention relates generally to plant molecular biology. In particular, the invention relates to compositions and methods for the regulation of plant growth and development through the modulation of either chorismate synthase or chorismate mutase gene expression or activity.
Chorismate is an essential substrate for the synthesis of p-aminobenzoate, folate, ubiquinone and the aromatic amino acids tryptophan, phenylalanine and tyrosine. Chorismate is produced in the seventh step of the shikimate pathway. This pathway has been described in numerous publications. See, for example, Shikimic Acid: Metabolism and Metabolites, John Wiley and Sons, Winchester, UK, 1993. The first four steps of the shikimate biosynthetic pathway lead to the production of shikimate. Shikimate is then converted to chorismate in next three steps of the pathway. First, shikimate is converted to shikimate-5-phosphate by shikimate kinase. Next, 3-enolpyruvateshikimate 5-phosphate synthase converts shikimate-5-phosphate to 5-enolpyruvylshikimate 3-phosphate, which is then converted to chorismate by the enzyme chorismate synthase.
The pathway leading to aromatic amino acid synthesis branches at chorismate. One branch leads to the synthesis of tryptophan. The other branch leads to the synthesis of phenylalanine and tyrosine. Thus, chorismate is the last common intermediate in the synthesis of tryptophan, phenylalanine and tyrosine.
In the branch leading to phenylalanine and tyrosine synthesis, chorismate is converted to prephenate by the enzyme chorismate mutase. Prephenate is the last common intermediate for biosyntheses of phenylalanine and tyrosine by two independent pathways that are present in both eukaryotes and prokaryotes.
Three isozymes of chorismate mutase, CM-1, CM-2 and CM-3 have been found in plants. Mobley et al. (1999) Gene 240:115-123. CM-1 and CM-3 are plastidic, while CM-2 is cytosolic. In Arabidopsis thaliana, CM-1 has 53% amino acid similarity with CM-2 and 68% amino acid similarity with CM-3.
The conversion of shikimate-5-phosphate to 5-enolpyruvylshikimate 3-phosphate is blocked by the commercially successful herbicide Roundup(trademark) (glyphosate). Accordingly, the shikimate pathway has been considered an attractive target for herbicides (PCT publication WO 00/05353, the contents of which are incorporated by reference, and Roberts et al. (1998) Nature 393:801-805). However, while it has been suggested that that chorismate synthase could be a candidate for a herbicide target (Bornemann et al. (1985) J Biol Chem 270:228111-22815), previous studies have not been able to ascertain whether chorismate synthase or chorismate mutase are essential for plant growth, which is a key parameter for determining potential herbicide targets. Nor are there any herbicides that are known to act my modifying the activity of either of these enzymes. Thus, it is necessary to determine whether such enzymes are critical to plant growth, before they can be considered useful targets in assays for the identification of herbicides and herbicide candidates.
The present inventors have discovered that chorismate mutase and chorismate synthase are essential for plant growth. Specifically, the inhibition of chorismate mutase or chorismate synthase gene expression in plant seedlings results in severe chlorosis, reduced growth and developmental abnormalities. Thus, in one aspect, the present invention provides compositions for the modulation of plant growth or development comprising chorismate synthase and chorismate mutase antisense and sense polynucleotides, dsRNA and ribozymes, and related expression cassettes and vectors. The compositions of the invention are particularly useful for the modulation and inhibition of plant growth. The invention further provides plants, plant cells, and seeds containing the polynucleotides of the invention.
The inventors have proven that chorismate synthase and chorismate mutase can be used as targets for the identification of herbicides. Thus, the present invention also provides methods for the identification of chemicals that modulate chorismate synthase and chorismate mutase biochemical reactions. The methods of the invention are useful for the identification of herbicides and for the inhibition of plant growth and development. In addition, the methods of the invention are useful for the identification of compounds that stimulate the expression or function of chorismate synthase or chorismate mutase expression or function. Such compounds can be used to promote or manipulate plant growth and development.